Inhaled corticosteroids (ICS) are the mainstay of asthma treatment, but evidence suggests a link between ICS usage and increased rates of respiratory infections. We assessed the composition of the ...asthmatic airways microbiome in asthma patients taking low and high dose ICS and the stability of the microbiome over a 2 week period.
We prospectively recruited 55 individuals with asthma. Of these, 22 were on low-dose ICS and 33 on high-dose ICS (16 on budesonide, 17 on fluticasone propionate). Sputum from each subject underwent DNA extraction, amplification and 16S rRNA gene sequencing of the bacterial component of the microbiome. 19 subjects returned for further sputum induction after 24 h and 2 weeks.
A total of 5,615,037 sequencing reads revealed 167 bacterial taxa in the asthmatic airway samples, with the most abundant being Streptococcus spp. No significant differences in sputum bacterial load or overall community composition were seen between the low- and high-dose ICS groups. However, Streptococcus spp. showed significantly higher relative abundance in subjects taking low-dose ICS (p = 0.002). Haemophilus parainfluenzae was significantly more abundant in subjects on high-dose fluticasone propionate than those on high-dose budesonide (p = 0.047). There were no statistically significant changes in microbiota composition over a 2-week period.
Whilst no significant differences were observed between the low- and high-dose ICS groups, increased abundance of the potential pathogen H. parainfluenzae was observed in patients taking high-dose fluticasone propionate compared to those taking high-dose budesonide. The microbiota were stable over fourteen days, providing novel evidence of the established community of bacteria in the asthmatic airways.
ClinicalTrials.gov NCT02671773.
Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases ...with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data.
We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation.
The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.
Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene ...targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.
Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several ...bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependent on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.
In this study, expression of GapA-1 was shown to be well conserved across diverse isolates of Neisseria species. Flow cytometry confirmed that GapA-1 could be detected on the cell surface, but only in a siaD-knockout (capsule-deficient) background, suggesting that GapA-1 is inaccessible to antibody in in vitro-grown encapsulated meningococci. The role of GapA-1 in meningococcal pathogenesis was addressed by mutational analysis and functional complementation. Loss of GapA-1 did not affect the growth of the bacterium in vitro. However, a GapA-1 deficient mutant showed a significant reduction in adhesion to human epithelial and endothelial cells compared to the wild-type and complemented mutant. A similar reduction in adhesion levels was also apparent between a siaD-deficient meningococcal strain and an isogenic siaD gapA-1 double mutant.
Our data demonstrates that meningococcal GapA-1 is a constitutively-expressed, highly-conserved surface-exposed protein which is antibody-accessible only in the absence of capsule. Mutation of GapA-1 does not affect the in vitro growth rate of N. meningitidis, but significantly affects the ability of the organism to adhere to human epithelial and endothelial cells in a capsule-independent process suggesting a role in the pathogenesis of meningococcal infection.
Fructose-1, 6-bisphosphate aldolases (FBA) are cytoplasmic glycolytic enzymes, which despite lacking identifiable secretion signals, have also been found localized to the surface of several bacteria ...where they bind host molecules and exhibit non-glycolytic functions. Neisseria meningitidis is an obligate human nasopharyngeal commensal, which has the capacity to cause life-threatening meningitis and septicemia. Recombinant native N. meningitidis FBA was purified and used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. Cell fractionation experiments showed that meningococcal FBA is localized both to the cytoplasm and the outer membrane. Flow cytometry demonstrated that outer membrane-localized FBA was surface-accessible to FBA-specific antibodies. Mutational analysis and functional complementation was used to identify additional functions of FBA. An FBA-deficient mutant was not affected in its ability to grow in vitro, but showed a significant reduction in adhesion to human brain microvascular endothelial and HEp-2 cells compared to its isogenic parent and its complemented derivative. In summary, FBA is a highly conserved, surface exposed protein that is required for optimal adhesion of meningococci to human cells.
Primary renal tubulointerstitial disease resulting from proximal tubule antigen-specific antibodies and immune complex formation has not been well characterized in humans. We report a cohort of ...patients with a distinct, underappreciated kidney disease characterized by kidney antibrush border antibodies and renal failure (ABBA disease). We identified ten patients with ABBA disease who had a combination of proximal tubule damage, IgG-positive immune deposits in the tubular basement membrane, and circulating antibodies reactive with normal human kidney proximal tubular brush border. All but one of the patients also had segmental glomerular deposits on renal biopsy specimen. Patients with ABBA disease were elderly and presented with AKI and subnephrotic proteinuria. Serum from all patients but not controls recognized a high molecular weight protein in renal tubular protein extracts that we identified as LDL receptor-related protein 2 (LRP2), also known as megalin, by immunoprecipitation and mass spectrometry. Immunostaining revealed that LRP2 specifically colocalized with IgG in the tubular immune deposits on the ABBA biopsy specimen but not the control specimen analyzed. Finally, ABBA serum samples but not control samples showed reactivity against recombinantly expressed N-terminal LRP2 fragments on Western blots and immunoprecipitated the recombinantly expressed N-terminal region of LRP2. This case series details the clinicopathologic findings of patients with ABBA disease and shows that the antigenic target of these autoantibodies is LRP2. Future studies are needed to determine the disease prevalence, stimulus for ABBA, and optimal treatment.
Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca 2+ sensitization/desensitization in smooth muscle. Ca 2+ sensitization induced by ...activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696
(of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that
elevate intracellular cGMP and cAMP promote Ca 2+ desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent
protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct
effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating
Thr-696. In vitro , phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle,
Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant
MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca 2+ sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism
of Ca 2+ desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents
phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.
Key points
Maternal shift work increases the risk of pregnancy complications, although its effects on progeny health after birth are not clear.
We evaluated the impact of a simulated shift work ...protocol for one‐third, two‐thirds or all of pregnancy on the metabolic health of sheep progeny.
Simulated shift work had no effect on growth, body size, body composition or glucose tolerance in pre‐pubertal or young adult progeny.
Glucose‐stimulated insulin secretion was reduced in adult female progeny and insulin sensitivity was increased in adult female singleton progeny.
The results of the present study do not support the hypothesis that maternal shift work exposure impairs metabolic health of progeny in altricial species.
Disrupted maternal circadian rhythms, such as those experienced during shift work, are associated with impaired progeny metabolism in rodents. The effects of disrupted maternal circadian rhythms on progeny metabolism have not been assessed in altricial, non‐litter bearing species. We therefore assessed postnatal growth from birth to adulthood, as well as body composition, glucose tolerance, insulin secretion and insulin sensitivity, in pre‐pubertal and young adult progeny of sheep exposed to control conditions (CON: 10 males, 10 females) or to a simulated shift work (SSW) protocol for the first one‐third (SSW0‐7: 11 males, 9 females), the first two‐thirds (SSW0‐14: 8 males, 11 females) or all (SSW0‐21: 8 males, 13 females) of pregnancy. Progeny growth did not differ between maternal treatments. In pre‐pubertal progeny (12–14 weeks of age), adiposity, glucose tolerance and insulin secretion during an i.v. glucose tolerance test and insulin sensitivity did not differ between maternal treatments. Similarly, in young adult progeny (12–14 months of age), food intake, adiposity and glucose tolerance did not differ between maternal treatments. At this age, however, insulin secretion in response to a glucose bolus was 30% lower in female progeny in the combined SSW groups compared to control females (P = 0.031), and insulin sensitivity of SSW0‐21 singleton females was 236% compared to that of CON singleton female progeny (P = 0.025). At least in this model, maternal SSW does not impair progeny metabolic health, with some evidence of greater insulin action in female young adult progeny.
Key points
Maternal shift work increases the risk of pregnancy complications, although its effects on progeny health after birth are not clear.
We evaluated the impact of a simulated shift work protocol for one‐third, two‐thirds or all of pregnancy on the metabolic health of sheep progeny.
Simulated shift work had no effect on growth, body size, body composition or glucose tolerance in pre‐pubertal or young adult progeny.
Glucose‐stimulated insulin secretion was reduced in adult female progeny and insulin sensitivity was increased in adult female singleton progeny.
The results of the present study do not support the hypothesis that maternal shift work exposure impairs metabolic health of progeny in altricial species.
Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using ...shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected.
Dominant clinically important species identified within the microbiomes included
,
and members of the
and
genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of
and high levels of
within some infants. We were able to demonstrate that in a third of the infants the
identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples.
We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.
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